Skin cancer is the most common form of human neoplasia and its incidence is rising rapidly. Many cancers affecting the skin including cutaneous T cell lymphomas (CTCL) exhibit silencing of tumor suppressor genes by DNA methylation. In this study, we propose to focus on the epigenetic silencing of selected tumor suppressor genes in two types of CTCL: mycosis fungoides (MF) and the Sezary syndrome (SS). We reported that the death receptor-ligand partners, FAS and FASL, are frequently expressed only weakly in MF/SS. We observed that FAS expression is transcriptionally regulated and that there is an inverse correlation between FAS expression and promoter methylation. We found that MF/SS is generally rich in DNMT1 (the DNA methyltransferase most likely responsible for epigenetic gene silencing) and that DNMT1 knockdown reverses FAS promoter methylation. We also discovered that folate antagonists like methotrexate (MTX) can inhibit DNA methylation by depleting S- adenosylmethionine (SAM), the main methyl donor for DNMTs. Our published data involving EMSA, supershift, ChIP and luciferase reporters show that the interaction of transcription factors (e.g. NFkB p50/p65) with the FAS promoter can be decreased by methylation resulting in less FAS expression. Using laser capture microdissection, pyrosequencing and multispectral imaging, we developed a method for quantitative gene analysis of methylation and expression (Q-GAME) in cells isolated from lesional skin. Using standard skin biopsies, Q-GAME can monitor in-situ the in-vivo effects of MTX and other agents on gene methylation and expression. With these findings and analytical tools in hand, we hypothesize that analogous to the situation with FAS, DNA methylation will account at least partially for decreased expression of FASL. Furthermore, MTX acting as a demethylator will reverse this gene repression and induce MF/SS apoptosis by enhancing activation of the FAS pathway. In Aim 1, we will use FAS/FASL-low MF/SS lines (HH, SZ4) that reflect the most common MF/SS clinical phenotype to optimize the demethylating effects of 5-aza, decitabine, MTX, MTX analogs and HDAC inhibitors on FAS and FASL in MF/SS. Inhibition of MTX and its analogs by exogenous SAM will be tested to confirm our proposed mechanism of action. We will validate these in-vitro studies ex-vivo using SS leukemic cells and determine the net effects on SS cell growth and survival. In Aim 2, we will explore the mechanisms regulating tumor suppressor gene silencing in FAS/FASL-low MF/SS lines. First we will compare the effects of DNMT1 inhibition to the effects of 5-aza because our preliminary data on DNMT1 knockdown indicate that it is a key mediator of gene methylation. Then we will use ChIP and co-immunoprecipitation to identify DNMT1 cofactors (e.g. STAT3, RelA, HDAC1) that bind to FAS and FASL. Finally, we will determine the impact of inhibition of these DNMT1 cofactors and upstream DNMT1 regulators on gene methylation. These in-vitro studies will be validated ex-vivo using SS leukemic cells. Targeting gene specific cofactors might allow relatively gene specific manipulation of DNA methylation. In Aim 3, to clinically validate our in-vitro and ex-vivo findings, we will use Q- GAME to quantitatively monitor FAS and FASL gene demethylation by MTX in-situ in MF/SS patient samples and determine the impact on gene expression and clinical outcome. We will use MTX because it is a standard MF/SS therapy and we have discovered that it is an effective demethylating agent. We will also assess the clinical effect of MTX in combination with other therapies (e.g. IFNa, HDAC inhibitors, UVB) that further enhance FAS or FASL by mechanisms different than MTX. In aggregate, these aims will identify and validate novel target genes for MF/SS therapy, provide strategies for modulating and monitoring their expression in the clinical setting, and determine their impact on clinical outcome. Successful completion of these highly translational aims depends on our extensive collection of MF/SS specimens and expertise with methods tailored specifically for this proposal.